Strain ORS571 Research Articles

Systematic Analysis of Lysine Acetylation Reveals Diverse Functions in Azorhizobium caulinodans Strain ORS571.

Protein acetylation can quickly modify the physiology of bacteria to respond to changes in environmental or nutritional conditions, but little information on these modifications is available in rhizobia. In this study, we report the lysine acetylome of Azorhizobium caulinodans strain ORS571, a model rhizobium isolated from stem nodules of the tropical legume Sesbania rostrata that is capable of fixing nitrogen in the free-living state and during symbiosis. Antibody enrichment and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were used to characterize the acetylome. There are 2,302 acetylation sites from 982 proteins, accounting for 20.8% of the total proteins. Analysis of the acetylated motifs showed the preferences for the amino acid residues around acetylated lysines. The response regulator CheY1, previously characterized to be involved in chemotaxis in strain ORS571, was identified as an acetylated protein, and a mutation of the acetylated site of CheY1 significantly impaired the strain's motility. In addition, a Zn+-dependent deacetylase (AZC_0414) was characterized, and the construction of a deletion mutant strain showed that it played a role in chemotaxis. Our study provides the first global analysis of lysine acetylation in ORS571, suggesting that acetylation plays a role in various physiological processes. In addition, we demonstrate its involvement in the chemotaxis process. The acetylome of ORS571 provides insights to investigate the regulation mechanism of rhizobial physiology. IMPORTANCE Acetylation is an important modification that regulates protein function and has been found to regulate physiological processes in various bacteria. The physiology of rhizobium A. caulinodans ORS571 is regulated by multiple mechanisms both when free living and in symbiosis with the host; however, the regulatory role of acetylation is not yet known. Here, we took an acetylome-wide approach to identify acetylated proteins in A. caulinodans ORS571 and performed clustering analyses. Acetylation of chemotaxis proteins was preliminarily investigated, and the upstream acetylation-regulating enzyme involved in chemotaxis was characterized. These findings provide new insights to explore the physiological mechanisms of rhizobia.

A glutamate synthase mutant of Bradyrhizobium sp. strain ORS285 is unable to induce nodules on Nod factor-independent Aeschynomene species

The Bradyrhizobium sp. strain ORS285 is able to establish a nitrogen-fixing symbiosis with both Nod factor (NF) dependent and NF-independent Aeschynomene species. Here, we have studied the growth characteristics and symbiotic interaction of a glutamate synthase (GOGAT; gltD::Tn5) mutant of Bradyrhizobium ORS285. We show that the ORS285 gltD::Tn5 mutant is unable to use ammonium, nitrate and many amino acids as nitrogen source for growth and is unable to fix nitrogen under free-living conditions. Moreover, on several nitrogen sources, the growth rate of the gltB::Tn5 mutant was faster and/or the production of the carotenoid spirilloxanthin was much higher as compared to the wild-type strain. The absence of GOGAT activity has a drastic impact on the symbiotic interaction with NF-independent Aeschynomene species. With these species, inoculation with the ORS285 gltD::Tn5 mutant does not result in the formation of nodules. In contrast, the ORS285 gltD::Tn5 mutant is capable to induce nodules on NF-dependent Aeschynomene species, but these nodules were ineffective for nitrogen fixation. Interestingly, in NF-dependent and NF-independent Aeschynomene species inoculation with the ORS285 gltD::Tn5 mutant results in browning of the plant tissue at the site of the infection suggesting that the mutant bacteria induce plant defence responses.

Bradyrhizobium sp. strain ORS278 promotes rice growth and its quorum sensing system is required for optimal root colonization.

Many Gram-negative bacteria communicate by using homoserine lactones (HSLs) as quorum sensing (QS) signals in a cell density-dependent manner. In addition to fatty acyl-HSL (acyl-HSL) signals, certain strains, most of them associated with plants, produce non-canonical aryl-HSLs such as cinnamoyl-HSL. However, the role of aryl-HSL in endophytic associations remained elusive. Bradyrhizobium sp. strain ORS278 possesses a LuxI-LuxR type QS system and produces cinnamoyl-HSL as a QS signal. Here, we report that strain ORS278 promotes growth of domesticated rice (Oryza sativa). QS mutants unable to produce cinnamoyl-HSL exhibited reduced plant-growth promoting activity in comparison to the parent strain ORS278. Likewise, the QS mutants were impaired in their ability to colonize rice roots. These findings suggest that genes controlled by cinnamoyl-HSL play an important role in the association between rice and ORS278. However, biofilm production was not visibly altered in these mutants. In conclusion, our study highlights the importance of aryl-HSLs in endophytic plant-bacteria interactions.

The Modification of the Flavonoid Naringenin by Bradyrhizobium sp. Strain ORS285 Changes the nod Genes Inducer Function to a Growth Stimulator.

As inducers of nodulation (nod) genes, flavonoids play an important role in the symbiotic interaction between rhizobia and legumes. However, in addition to the control of expression of nod genes, many other effects of flavonoids on rhizobial cells have been described. Here, we show that the flavonoid naringenin stimulates the growth of the photosynthetic Bradyrhizobium sp. strain ORS285. This growth-stimulating effect was still observed for strain ORS285 with nodD1, nodD2, or the naringenin-degrading fde operon deleted. Phenotypic microarray analysis indicates that in cells grown in the presence of naringenin, the glycerol and fatty acid metabolism is activated. Moreover, electron microscopic and enzymatic analyses show that polyhydroxy alkanoate metabolism is altered in cells grown in the presence of naringenin. Although strain ORS285 was able to degrade naringenin, a fraction was converted into an intensely yellow-colored molecule with an m/z (+) of 363.0716. Further analysis indicates that this molecule is a hydroxylated and O-methylated form of naringenin. In contrast to naringenin, this derivative did not induce nod gene expression, but it did stimulate the growth of strain ORS285. We hypothesize that the growth stimulation and metabolic changes induced by naringenin are part of a mechanism to facilitate the colonization and infection of naringenin-exuding host plants.

Inactivation of the Phosphatase CheZ Alters Cell-Surface Properties of Azorhizobium caulinodans ORS571 and Symbiotic Association with Sesbania rostrata.

Azorhizobium caulinodans can form root and stem nodules with the host plant Sesbania rostrata. The role of the CheZ phosphatase in the A. caulinodans chemotaxis pathway was previously explored using the nonchemotactic cheZ mutant strain (AC601). This mutant displayed stronger attachment to the root surface, enhancing early colonization; however, this did not result in increased nodulation efficiency. In this study, we further investigated the role of CheZ in the interaction between strain ORS571 and the roots of its host plant. By tracking long-term colonization dynamic of cheZ mutant marked with LacZ, we found a decrease of colonization of the cheZ mutant during this process. Furthermore, the cheZ mutant could not spread on the root surface freely and was gradually outcompeted by the wild type in original colonization sites. Quantitative reverse-transcription PCR analyses showed that exp genes encoding exopolysaccharides synthesis, including oac3, were highly expressed in the cheZ mutant. Construction of a strain carrying a deletion of both cheZ and oac3 resulted in a mutant strain defective in the colonization process to the same extent as found with the oac3 single-mutant strain. This result suggested that the enhanced colonization of the cheZ mutant may be achieved through regulating the formation of exopolysaccharides. This shows the importance of the chemotactic proteins in the interaction between rhizobia and host plants, and expands our understanding of the symbiosis interaction between rhizobium and host plant.

Transcriptomic dissection of Bradyrhizobium sp. strain ORS285 in symbiosis with Aeschynomene spp. inducing different bacteroid morphotypes with contrasted symbiotic efficiency.

To circumvent the paucity of nitrogen sources in the soil legume plants establish a symbiotic interaction with nitrogen-fixing soil bacteria called rhizobia. During symbiosis, the plants form root organs called nodules, where bacteria are housed intracellularly and become active nitrogen fixers known as bacteroids. Depending on their host plant, bacteroids can adopt different morphotypes, being either unmodified (U), elongated (E) or spherical (S). E- and S-type bacteroids undergo a terminal differentiation leading to irreversible morphological changes and DNA endoreduplication. Previous studies suggest that differentiated bacteroids display an increased symbiotic efficiency (E > U and S > U). In this study, we used a combination of Aeschynomene species inducing E- or S-type bacteroids in symbiosis with Bradyrhizobium sp. ORS285 to show that S-type bacteroids present a better symbiotic efficiency than E-type bacteroids. We performed a transcriptomic analysis on E- and S-type bacteroids formed by Aeschynomene afraspera and Aeschynomene indica nodules and identified the bacterial functions activated in bacteroids and specific to each bacteroid type. Extending the expression analysis in E- and S-type bacteroids in other Aeschynomene species by qRT-PCR on selected genes from the transcriptome analysis narrowed down the set of bacteroid morphotype-specific genes. Functional analysis of a selected subset of 31 bacteroid-induced or morphotype-specific genes revealed no symbiotic phenotypes in the mutants. This highlights the robustness of the symbiotic program but could also indicate that the bacterial response to the plant environment is partially anticipatory or even maladaptive. Our analysis confirms the correlation between differentiation and efficiency of the bacteroids and provides a framework for the identification of bacterial functions that affect the efficiency of bacteroids.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

A cheZ-Like Gene in Azorhizobium caulinodans Is a Key Gene in the Control of Chemotaxis and Colonization of the Host Plant.

Chemotaxis can provide bacteria with competitive advantages for survival in complex environments. The CheZ chemotaxis protein is a phosphatase, affecting the flagellar motor in Escherichia coli by dephosphorylating the response regulator phosphorylated CheY protein (CheY∼P) responsible for clockwise rotation. A cheZ gene has been found in Azorhizobium caulinodans ORS571, in contrast to other rhizobial species studied so far. The CheZ protein in strain ORS571 has a conserved motif similar to that corresponding to the phosphatase active site in E. coli The construction of a cheZ deletion mutant strain and of cheZ mutant strains carrying a mutation in residues of the putative phosphatase active site showed that strain ORS571 participates in chemotaxis and motility, causing a hyperreversal behavior. In addition, the properties of the cheZ deletion mutant revealed that ORS571 CheZ is involved in other physiological processes, since it displayed increased flocculation, biofilm formation, exopolysaccharide (EPS) production, and host root colonization. In particular, it was observed that the expression of several exp genes, involved in EPS synthesis, was upregulated in the cheZ mutant compared to that in the wild type, suggesting that CheZ negatively controls exp gene expression through an unknown mechanism. It is proposed that CheZ influences the Azorhizobium-plant association by negatively regulating early colonization via the regulation of EPS production. This report established that CheZ in A. caulinodans plays roles in chemotaxis and the symbiotic association with the host plant.IMPORTANCE Chemotaxis allows bacteria to swim toward plant roots and is beneficial to the establishment of various plant-microbe associations. The level of CheY phosphorylation (CheY∼P) is central to the chemotaxis signal transduction. The mechanism of the signal termination of CheY∼P remains poorly characterized among Alphaproteobacteria, except for Sinorhizobium meliloti, which does not contain CheZ but which controls CheY∼P dephosphorylation through a phosphate sink mechanism. Azorhizobium caulinodans ORS571, a microsymbiont of Sesbania rostrata, has an orphan cheZ gene besides two cheY genes similar to those in S. meliloti In addition to controlling the chemotaxis response, the CheZ-like protein in strain ORS571 is playing a role by decreasing bacterial adhesion to the host plant, in contrast to the general situation where chemotaxis-associated proteins promote adhesion. In this study, we identified a CheZ-like protein among Alphaproteobacteria functioning in chemotaxis and the A. caulinodans-S. rostrata symbiosis.

The infection and impact of Azorhizobium caulinodans ORS571 on wheat (Triticum aestivum L.)

Based on our previous study, cereal crop wheat (Triticum aestivum L.) could be infected by rhizobia Azorhizobium caulinodans ORS571, and form para-nodules with the induction of 2.4-dichlorophenoxyacetic acid, a common plant growth regulator. To enhance this infection and the potential agricultural application, we compared six different infection methods (Direct seed dip; Seed germination dip; Pruned-root dip; Foliar spray; Circum-soil dip; Seed dip and circum-soil dip) for achieving the high efficient infection of A. caulinodans into wheat plants by employing a green fluorescent protein (gfp)-labeled Azorhizobium caulinodans strain ORS571. With proper methods, copious rhizobia could enter the interior and promote the growth of wheat to the hilt. Circum-soil dip was proved to be the most efficient method, seed germination dip and pruned-root dip is the last recommended to infect wheat, seed germination dip and seed dip and circum-soil dip showed better effects on plant growth, pruned-root dip did not show too much effect on plant growth. This study laid the foundation for understanding the interaction between rhizobia and cereal crops and the growth-promoting function of rhizobia.

The Very Long Chain Fatty Acid (C26:25OH) Linked to the Lipid A Is Important for the Fitness of the Photosynthetic Bradyrhizobium Strain ORS278 and the Establishment of a Successful Symbiosis with Aeschynomene Legumes.

In rhizobium strains, the lipid A is modified by the addition of a very long-chain fatty acid (VLCFA) shown to play an important role in rigidification of the outer membrane, thereby facilitating their dual life cycle, outside and inside the plant. In Bradyrhizobium strains, the lipid A is more complex with the presence of at least two VLCFAs, one covalently linked to a hopanoid molecule, but the importance of these modifications is not well-understood. In this study, we identified a cluster of VLCFA genes in the photosynthetic Bradyrhizobium strain ORS278, which nodulates Aeschynomene plants in a Nod factor-independent process. We tried to mutate the different genes of the VLCFA gene cluster to prevent the synthesis of the VLCFAs, but only one mutant in the lpxXL gene encoding an acyltransferase was obtained. Structural analysis of the lipid A showed that LpxXL is involved in the transfer of the C26:25OH VLCFA to the lipid A but not in the one of the C30:29OH VLCFA which harbors the hopanoid molecule. Despite maintaining the second VLCFA, the ability of the mutant to cope with various stresses (low pH, high temperature, high osmolarity, and antimicrobial peptides) and to establish an efficient nitrogen-fixing symbiosis was drastically reduced. In parallel, we investigated whether the BRADO0045 gene, which encodes a putative acyltransferase displaying a weak identity with the apo-lipoprotein N-acyltransferase Lnt, could be involved in the transfer of the C30:29OH VLCFA to the lipid A. Although the mutant exhibited phenotypes similar to the lpxXL mutant, no difference in the lipid A structure was observed from that in the wild-type strain, indicating that this gene is not involved in the modification of lipid A. Our results advance our knowledge of the biosynthesis pathway and the role of VLCFAs-modified lipid A in free-living and symbiotic states of Bradyrhizobium strains.

Complete Genome Sequence of Bradyrhizobium sp. ORS285, a Photosynthetic Strain Able To Establish Nod Factor-Dependent or Nod Factor-Independent Symbiosis with Aeschynomene Legumes.

ABSTRACTHere, we report the complete genome sequence of Bradyrhizobium sp. strain ORS285, which is able to nodulate Aeschynomene legumes using two distinct strategies that differ in the requirement of Nod factors. The genome sequence information of this strain will help understanding of the different mechanisms of interaction of rhizobia with legumes.

The role of rhizobial (NifV) and plant (FEN1) homocitrate synthases in Aeschynomene/photosynthetic Bradyrhizobium symbiosis

In the most studied rhizobium-legume interactions, the host plant supplies the symbiont with homocitrate, an essential co-factor of the nitrogenase enzyme complex, via the expression of a nodule-specific homocitrate synthase FEN1. Photosynthetic bradyrhizobia interacting with Nod factor (NF) dependent and NF-independent Aeschynomene legumes are able to synthesize homocitrate themselves as they contain a nifV gene encoding a homocitrate synthase. Here, we show that in the model strain ORS285, nifV is required for free-living and symbiotic dinitrogen fixation with NF-independent Aeschynomene species. In contrast, in symbiosis with NF-dependent Aeschynomene species, the nifV requirement for efficient nitrogen fixation was found to be host plant dependent. Interestingly, orthologs of FEN1 were found in both NF-dependent and NF-independent Aeschynomene species. However, a high nodule specific induction of FEN1 expression was only observed in A. afraspera, a host plant in which nifV is not required for symbiotic dinitrogen fixation. These data indicate that efficient symbiotic nitrogen fixation in many of the tested Aeschynomene species requires rhizobial homocitrate synthesis. Considering that more than 10% of the fully sequenced rhizobium strains do contain a nifV gene, the Aeschynomene/photosynthetic Bradyrhizobium interaction is likely not the only rhizobium/legume symbiosis where rhizobial nifV expression is required.

NodD1 and NodD2 Are Not Required for the Symbiotic Interaction of Bradyrhizobium ORS285 with Nod-Factor-Independent Aeschynomene Legumes.

Photosynthetic Bradyrhizobium strain ORS285 forms nitrogen-fixing nodules on the roots and stems of tropical aquatic legumes of the Aeschynomene genus. Depending on the Aeschynomene species, this symbiotic interaction does or does not rely on the synthesis of Nod-factors (NFs). However, whether during the interaction of Bradyrhizobium ORS285 with NF-independent Aeschynomene species the nod genes are expressed and if the general regulator NodD plays a symbiotic role is unknown. Expression studies showed that in contrast to the interaction with the NF-dependent Aeschynomene species, A. afraspera, the Bradyrhizobium ORS285 nod genes are not induced upon contact with the NF-independent host plant A. indica. Mutational analysis of the two nodD genes present in ORS285, showed that deletion of nodD1 and nodD2 did not affect the symbiotic interaction between Bradyrhizobium ORS285 and A. indica whereas the deletions had an effect on the symbiotic interaction with A. afraspera plants. In addition, when the expression of nod genes was artificially induced by adding naringenin to the plant growth medium, the nodulation of A. indica by Bradyrhizobium ORS285 is delayed and resulted in lower nodule numbers.

Preferential association of endophytic bradyrhizobia with different rice cultivars and its implications for rice endophyte evolution.

Plant colonization by bradyrhizobia is found not only in leguminous plants but also in nonleguminous species such as rice. To understand the evolution of the endophytic symbiosis of bradyrhizobia, the effect of the ecosystems of rice plantations on their associations was investigated. Samples were collected from various rice (Oryza sativa) tissues and crop rotational systems. The rice endophytic bradyrhizobia were isolated on the basis of oligotrophic properties, selective medium, and nodulation on siratro (Macroptilium atropurpureum). Six bradyrhizobial strains were obtained exclusively from rice grown in a crop rotational system. The isolates were separated into photosynthetic bradyrhizobia (PB) and nonphotosynthetic bradyrhizobia (non-PB). Thai bradyrhizobial strains promoted rice growth of Thai rice cultivars better than the Japanese bradyrhizobial strains. This implies that the rice cultivars possess characteristics that govern rice-bacterium associations. To examine whether leguminous plants in a rice plantation system support the persistence of rice endophytic bradyrhizobia, isolates were tested for legume nodulation. All PB strains formed symbioses with Aeschynomene indica and Aeschynomene evenia. On the other hand, non-PB strains were able to nodulate Aeschynomene americana, Vigna radiata, and M. atropurpureum but unable to nodulate either A. indica or A. evenia. Interestingly, the nodABC genes of all of these bradyrhizobial strains seem to exhibit low levels of similarity to those of Bradyrhizobium diazoefficiens USDA110 and Bradyrhizobium sp. strain ORS285. From these results, we discuss the evolution of the plant-bradyrhizobium association, including nonlegumes, in terms of photosynthetic lifestyle and nod-independent interactions.

Rhizobial Synthesized Cytokinins Contribute to But Are Not Essential for the Symbiotic Interaction Between Photosynthetic Bradyrhizobia and Aeschynomene Legumes

Cytokinins (CK) play an important role in the formation of nitrogen-fixing root nodules. It has been known for years that rhizobia secrete CK in the extracellular medium but whether they play a role in nodule formation is not known. We have examined this question using the photosynthetic Bradyrhizobium sp. strain ORS285 which is able to nodulate Aeschynomene afraspera and A. indica using a Nod-dependent or Nod-independent symbiotic process, respectively. CK profiling showed that the most abundant CK secreted by Bradyrhizobium sp. strain ORS285 are the 2MeS (2-methylthiol) derivatives of trans-zeatin and isopentenyladenine. In their pure form, these CK can activate legume CK receptors in vitro, and their exogenous addition induced nodule-like structures on host plants. Deletion of the miaA gene showed that transfer RNA degradation is the source of CK production in Bradyrhizobium sp. strain ORS285. In nodulation studies performed with A. indica and A. afraspera, the miaA mutant had a 1-day delay in nodulation and nitrogen fixation. Moreover, A. indica plants formed considerably smaller but more abundant nodules when inoculated with the miaA mutant. These data show that CK produced by Bradyrhizobium sp. strain ORS285 are not the key signal triggering nodule formation during the Nod-independent symbiosis but they contribute positively to nodule development in Aeschynomene plants.

Aeschynomene evenia, a Model Plant for Studying the Molecular Genetics of the Nod-Independent Rhizobium-Legume Symbiosis

Research on the nitrogen-fixing symbiosis has been focused, thus far, on two model legumes, Medicago truncatula and Lotus japonicus, which use a sophisticated infection process involving infection thread formation. However, in 25% of the legumes, the bacterial entry occurs more simply in an intercellular fashion. Among them, some Aeschynomene spp. are nodulated by photosynthetic Bradyrhizobium spp. that do not produce Nod factors. This interaction is believed to represent a living testimony of the ancestral state of the rhizobium-legume symbiosis. To decipher the mechanisms of this Nod-independent process, we propose Aeschynomene evenia as a model legume because it presents all the characteristics required for genetic and molecular analysis. It is a short-perennial and autogamous species, with a diploid and relatively small genome (2n=20; 460 Mb/1C). A. evenia 'IRFL6945' is nodulated by the well-characterized photosynthetic Bradyrhizobium sp. strain ORS278 and is efficiently transformed by Agrobacterium rhizogenes. Aeschynomene evenia is genetically homozygous but polymorphic accessions were found. A manual hybridization procedure has been set up, allowing directed crosses. Therefore, it should be relatively straightforward to unravel the molecular determinants of the Nod-independent process in A. evenia. This should shed new light on the evolution of rhizobium-legume symbiosis and could have important agronomic implications.

Comparative Genomics of Aeschynomene Symbionts: Insights into the Ecological Lifestyle of Nod-Independent Photosynthetic Bradyrhizobia

Tropical aquatic species of the legume genus Aeschynomene are stem- and root-nodulated by bradyrhizobia strains that exhibit atypical features such as photosynthetic capacities or the use of a nod gene-dependent (ND) or a nod gene-independent (NI) pathway to enter into symbiosis with legumes. In this study we used a comparative genomics approach on nine Aeschynomene symbionts representative of their phylogenetic diversity. We produced draft genomes of bradyrhizobial strains representing different phenotypes: five NI photosynthetic strains (STM3809, ORS375, STM3847, STM4509 and STM4523) in addition to the previously sequenced ORS278 and BTAi1 genomes, one photosynthetic strain ORS285 hosting both ND and NI symbiotic systems, and one NI non-photosynthetic strain (STM3843). Comparative genomics allowed us to infer the core, pan and dispensable genomes of Aeschynomene bradyrhizobia, and to detect specific genes and their location in Genomic Islands (GI). Specific gene sets linked to photosynthetic and NI/ND abilities were identified, and are currently being studied in functional analyses.

PhotosyntheticBradyrhizobiumSp. Strain ORS285 Synthesizes 2-O-Methylfucosylated Lipochitooligosaccharides fornodGene–Dependent Interaction withAeschynomenePlants

Bradyrhizobium sp. strain ORS285 is a photosynthetic bacterium that forms nitrogen-fixing nodules on the roots and stems of tropical aquatic legumes of the Aeschynomene genus. The symbiotic interaction of Bradyrhizobium sp. strain ORS285 with certain Aeschynomene spp. depends on the presence of nodulation (nod) genes whereas the interaction with other species is nod gene independent. To study the nod gene-dependent molecular dialogue between Bradyrhizobium sp. strain ORS285 and Aeschynomene spp., we used a nodB-lacZ reporter strain to monitor the nod gene expression with various flavonoids. The flavanones liquiritigenin and naringenin were found to be the strongest inducers of nod gene expression. Chemical analysis of the culture supernatant of cells grown in the presence of naringenin showed that the major Nod factor produced by Bradyrhizobium sp. strain ORS285 is a modified chitin pentasaccharide molecule with a terminal N-C(18:1)-glucosamine and with a 2-O-methyl fucose linked to C-6 of the reducing glucosamine. In this respect, the Bradyrhizobium sp. strain ORS285 Nod factor is the same as the major Nod factor produced by the nonphotosynthetic Bradyrhizobium japonicum USDA110 that nodulates the roots of soybean. This suggests a classic nod gene-dependent molecular dialogue between Bradyrhizobium sp. strain ORS285 and certain Aeschynomene spp. This is supported by the fact that B. japonicum USDA110 is able to form N(2)-fixing nodules on both the roots and stems of Aeschynomene afraspera.

Nodulation of Aeschynomene afraspera and A. indica by Photosynthetic Bradyrhizobium Sp. Strain ORS285: The Nod-Dependent Versus the Nod-Independent Symbiotic Interaction

Here, we present a comparative analysis of the nodulation processes of Aeschynomene afraspera and A. indica that differ in their requirement for Nod factors (NF) to initiate symbiosis with photosynthetic bradyrhizobia. The infection process and nodule organogenesis was examined using the green fluorescent protein-labeled Bradyrhizobium sp. strain ORS285 able to nodulate both species. In A. indica, when the NF-independent strategy is used, bacteria penetrated the root intercellularly between axillary root hairs and invaded the subepidermal cortical cells by invagination of the host cell wall. Whereas the first infected cortical cells collapsed, the infected ones immediately beneath kept their integrity and divided repeatedly to form the nodule. In A. afraspera, when the NF-dependent strategy is used, bacteria entered the plant through epidermal fissures generated by the emergence of lateral roots and spread deeper intercellularly in the root cortex, infecting some cortical cells during their progression. Whereas the infected cells of the lower cortical layers divided rapidly to form the nodule, the infected cells of the upper layers gave rise to an outgrowth in which the bacteria remained enclosed in large tubular structures. Together, two distinct modes of infection and nodule organogenesis coexist in Aeschynomene legumes, each displaying original features.

Large-Scale Transposon Mutagenesis of Photosynthetic Bradyrhizobium Sp. Strain ORS278 Reveals New Genetic Loci Putatively Important for Nod-Independent Symbiosis with Aeschynomene indica

Photosynthetic Bradyrhizobium strains possess the unusual ability to form nitrogen-fixing nodules on a specific group of legumes in the absence of Nod factors. To obtain insight into the bacterial genes involved in this Nod-independent symbiosis, we screened 15,648 Tn5 mutants of Bradyrhizobium sp. strain ORS278 for clones affected in root symbiosis with Aeschynomene indica. From the 268 isolated mutants, 120 mutants were altered in nodule development (Ndv(-)) and 148 mutants were found to be deficient in nitrogen fixation (Fix(-)). More than 50% of the Ndv(-) mutants were found to be altered in purine biosynthesis, strengthening the previous hypothesis of a symbiotic role of a bacterial purine derivative during the Nod-independent symbiosis. The other Ndv(-) mutants were auxotrophic for pyrimidines and amino acids (leucine, glutamate, and lysine) or impaired in genes encoding proteins of unknown function. The Fix(-) mutants were found to be affected in a wide variety of cellular processes, including both novel (n = 56) and previously identified (n = 31) genes important in symbiosis. Among the novel genes identified, several were involved in the Calvin cycle, suggesting that CO(2) fixation could play an important role during this symbiosis.

Insights learned from pBTAi1, a 229-kb accessory plasmid from Bradyrhizobium sp. strain BTAi1 and prevalence of accessory plasmids in other Bradyrhizobium sp. strains

In silico, physiological and in planta analyses were used to characterize pBTAi1, a 229-kb accessory plasmid from Bradyrhizobium sp. strain BTAi1, and assess its potential ecological function under free-living and symbiotic growth conditions. Sequence analysis revealed the presence of an uptake hydrogenase system, a repABC family plasmid replication module and open reading frames encoding type IV secretion system, TraI and TraR autoinducer proteins and several copper resistance-related proteins. Bradyrhizobium sp. BTAi1 was capable of growing in 200 mg l(-1) CuCl2. In contrast, the closely related, plasmid-free Bradyrhizobium sp. strain ORS278 could not grow at copper concentrations exceeding 100 mg l(-1). The plasmid-localized hydrogenase genes were phylogenetically distinct from those typically found in other rhizobial species, and were most related to hup genes from Thiobacillus denitrificans. The induction of the plasmid-borne hydrogenase genes during symbiosis was significantly lower than the two chromosomal-borne hydrogenase clusters. CHEF-pulsed-field gel electrophoresis was used for a comprehensive analysis of the diversity, abundance and genetic composition of accessory plasmids in other Bradyrhizobium strains. Plasmids were detected in 11 of 46 (23.9%) geographically diverse Bradyrhizobium japonicum and Bradyrhizobium elkanii strains, isolated from the United States, China and Thailand. Plasmid size was heterogeneous, ranging from 75 to 330 kb, with only two strains (DASA01244 and DASA01265) harboring plasmids with identical (240 kb) size. None of the plasmids harbored nodulation or hydrogenase genes. Taken together, our results indicate that while plasmids having ecologically significant functions may be detected in Bradyrhizobium sp. strains, they lack genes necessary for symbioses with legumes.